Our aims during this grant period will be to delineate further the signals, effectors, pathways and rate-limiting biochemical steps for both the acute and adaptive changes in renal ammoniagenesis in response to alterations in H+ and K+ homeostasis. In addition the mechanisms of action of several modulators of ammoniagenesis will be explored, including prostaglandins, calcium, cyclic AMP, glucocorticoids and aldosterone. The major experimental model utilized will be renal epithelium grown in cell culture. The LLC-PK1 continuous pig cell line will be used. In addition primary cultures of rat, dog and human proximal tubular epithelium will be developed and utilized. Techniques employed with cultured cells will include enzyme analysis, measurments of metabolites, pulse chase isotopic studies, investigation of isolated mitochondria, and studies of the molecular biology of adaptation. In addition to cell culture, experiments with intact animals, isolated perfused kidney, renal cortical tubule and isolated mitochondria will be used to address the mechanisms of decreased NH3 production with acute alkalosis, the signal for adaptation to chronic acidosis, the inhibitory effect of PGF2alpha and the K+ sparing effect of increased NH3 production.